Journal: Diagnostics

Article Title: Performance of Antigen Detection Tests for SARS-CoV-2: A Systematic Review and Meta-Analysis

doi: 10.3390/diagnostics12061388

Figure Lengend Snippet: Characteristics of the 235 studies included in the meta-analysis.

Article Snippet: Krüger et al. [ ] , Germany/UK , N , nsp/ts , 1. FIA 2. LFIA 3. LFIA/virus culture data , 1. 2019-nCoV Ag Fluorescence Rapid Test Kit 2. COVID-19 Ag Respi-Strip 3. STANDARD Q COVID-19 Ag Test , 1. Shenzhen Bioeasy Biotechnology Co. Ltd., Guangdong Province, China 2. Coris Bioconcept, Gembloux, Belgium 3. SD Biosensor, Inc., Gyeonggi-do, Korea , Up to 30/up to 40/30–40 , 1. Rapid/detector 2. Rapid 3. Rapid , 2407 , 72 , 2335.

Techniques: Diagnostic Assay, Surround Optical-fiber Immunoassay, Fluorescence, Enzyme-linked Immunosorbent Assay, Raman Spectroscopy, Antigen Assay, Mass Spectrometry, Stripping Membranes

Journal: Applied microbiology and biotechnology

Article Title: Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

doi: 10.1007/s00253-022-12017-7

Figure Lengend Snippet: Fig. 4 Impact of spacer length on cutinase-GFP11 secretion with B. subtilis signal peptides NprE, Pel, Epr, and Bsn. C. glu- tamicum (a–d) or B. subtilis (e–h) were used as secretion hosts. Cutinase-GFP11 was detected in supernatant samples of C. glutamicum by cutinase activity and split GFP assay in analytical duplicates of three cultivation supernatants, respectively. Cutinase-GFP11 in B. subtilis cultivation superna- tants was detected in triplicates. Enzymatic activity and holo- GFP fluorescence after satura- tion were normalized to x-fold changes relative to the respec- tive strain with a 4 nt ribosome binding site spacer (dashed line). The Y-axis scaling for subfigure e differs from the oth- ers and is highlighted in green. *data from Volkenborn et al. (2020), Creative Commons CC BY license, see Reprints and Permissions

Article Snippet: Monitoring cutinase secretion using the C. glutamicum ATCC 13032 K9 biosensor strain Cutinase secretion experiments were performed using C. glutamicum ATCC 13032 K9 biosensor cells that enable the monitoring of recombinant protein secretion by means of cellular fluorescence (Jurischka et al. 2020).

Techniques: Activity Assay, Split GFP Assay, Fluorescence, Binding Assay

Journal: Applied microbiology and biotechnology

Article Title: Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

doi: 10.1007/s00253-022-12017-7

Figure Lengend Snippet: Fig. 7 Secretion performance and secretion stress associated with cutinase-GFP11 secretion using different signal peptides in combination with an 8 nt ribosome binding site spacer. Secreted cutinase-GFP11 from C. glutamicum K9 pCMEx8- [SP]-Cutinase was detected via split GFP assay and cutinase activity measurements that were performed manually (a). At the end of the cultivation, the cell- specific eYFP fluorescence was determined of two independent clones and the average is shown with standard deviation (b). The dashed line indicates the normal background fluorescence associ- ated with secretion stress in the absence of cutinase expression

Article Snippet: Monitoring cutinase secretion using the C. glutamicum ATCC 13032 K9 biosensor strain Cutinase secretion experiments were performed using C. glutamicum ATCC 13032 K9 biosensor cells that enable the monitoring of recombinant protein secretion by means of cellular fluorescence (Jurischka et al. 2020).

Techniques: Binding Assay, Split GFP Assay, Activity Assay, Fluorescence, Clone Assay, Standard Deviation, Expressing

Journal: Biosensors

Article Title: Point-of-Care Diagnostics for Farm Animal Diseases: From Biosensors to Integrated Lab-on-Chip Devices

doi: 10.3390/bios12070455

Figure Lengend Snippet: Available electrochemical and optical biosensors for the detection of mastitis and animal diseases. The available literature for the remaining transducing options is included in the main text.

Article Snippet: Swine-origin influenza A H1N1 virus , Anti-H1 antibody (ProSci, Poway, CA, USA) , SPR chip (BK7 glass slide coated with a laminated Ag/Au 37/8 nm, metal layer), paired-surface plasma-wave biosensor , Surface plasmon resonance (SPR) , Mimic solution (human mucosa in PBS) , Theoretical LOD of 30 PFU/mL, 1.8 × 10 2 PFU/mL. Detection in 20 min , [ ] .

Techniques: Impedance Spectroscopy, Bioprocessing, Produced, Virus, Magnetic Beads, Affinity Chromatography, Purification, Avidin-Biotin Assay, Recombinant, Irradiation, Biomarker Discovery, Electrochemiluminescence, Bacteria, Clinical Proteomics, SPR Assay, Circular Dichroism, Labeling, Fluorescence, Spectrophotometry, Förster Resonance Energy Transfer, Modification, Membrane, Imaging, Refractive Index

Journal: Nature Communications

Article Title: Dynamic interactions and Ca 2+ -binding modulate the holdase-type chaperone activity of S100B preventing tau aggregation and seeding

doi: 10.1038/s41467-021-26584-2

Figure Lengend Snippet: a , b Representative immunofluorescence images of tau RD P301S FRET biosensor cells exposed to tau oligomers (0.1 µM and 0.5 µM) alone ( a ) or to tau oligomers preincubated with S100B (5 µM) ( b ) for 24 h in the presence of lipofectamine. In all, 5 µM S100B data are shown as representative data of the effect of S100B alone in tau biosensor cells, as even higher concentrations did not cause seeding. Cell nuclei stained with DAPI (blue). Objective used: 40X; scale bar 50 μm. c Statistical analysis with data presented as mean and standard error of mean from n ≥ 3 independent experimental replicates using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. Specifically, for assays with vehicle and 5 µM S100B n = 3, for assays with 0.1 µM TauO n = 5, for assays with 0.5 µM TauO n = 4, and for assays with 5 µM S100B + 0.1 µM TauO and 5 µM S100B + 0.5 µM TauO n = 6. Statistical significance at p < 0.05 was considered, **** p < 0.0001 and *** p = 0.0009.

Article Snippet: The seeding assay of tau oligomers (TauO), S100B and TauO–S100B complex, was carried out in Tau RD P301S CFP/YFP biosensor cells (ATCC, Cat. CRL-3275) as described previously .

Techniques: Immunofluorescence, Staining, Comparison

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Overview of the workflow—B cells (or enriched ASCs) are isolated from mice (bone marrow or spleen) or human PBMCs. Cells are mixed with liquid BG-agarose at 37 °C and encapsulated into picolitre water-in-oil emulsion droplets using a flow-focusing junction. Droplets are collected on ice for agarose gelation and demulsified, creating stable hydrogel beads around each cell. The BG-agarose is converted into an antibody capture matrix by the addition of recombinant capture reagents that are fused to the SNAP-tag, an enzyme that reacts with BG moieties. During incubation, antibodies secreted by a single cell are captured in the hydrogel surrounding the cell. Cells that have secreted antigen-specific antibodies are identified with fluorescently labeled detection reagents (antigens, secondary antibodies and antibodies against cell-surface markers), sorted using flow cytometry and sequenced. Antibody sequences can be obtained within 4 days, and recombinant antibodies for testing are generated in 2 weeks. b , Agarose-based antibody capture matrix. Agarose is chemically modified to contain BG moieties that react covalently with the SNAP-tag. Single-domain antibodies (VHHs) against the constant region of antibody light chains are expressed as SNAP-tag fusions and immobilized in the BG-agarose hydrogel, creating the capture matrix. c , Antibody capture by BG-agarose hydrogel beads functionalized with VHH–SNAP. Antibody capture (anti-streptavidin mouse IgG) and antigen binding (streptavidin–GFP) were analyzed by flow cytometry. The plot shows at least 220 events per condition at a 5% contour level. d , Antibody secretion by single OVA-specific mouse bone marrow plasma cells. Representative confocal microscopy image from two independent experiments showing a single cell encapsulated in VHH-functionalized BG-agarose stained with fluorescently labeled OVA (AF555), anti-CD138 antibodies (AF647) and anti-mouse IgG antibodies (AF405). e , Sorting of OVA-specific mouse bone marrow plasma cells. Hydrogel beads containing plasma cells that secreted OVA-specific IgG were sorted by FACS (gated as live/FLAG + /CD138 + /IgM − /IgG + ). The plots show 10,168 (CD138 + ) and 411 (IgG + ) events at 2% contour level. f , Characteristics of mouse anti-OVA antibodies—variable domain genes (V and J), third complementarity-determining region amino acid sequences (CDR3s) and equilibrium dissociation constants ( K D ).

Article Snippet: For in-tandem assays of mouse antibodies, biotinylated RBD (10 μg ml −1 ) was immobilized on streptavidin BLI biosensors (Sartorius) for 300 s. The biosensors were then probed with 100 nM of the first antibody for 600 s followed by 100 nM of the second antibody for 300 s. Independent binding of both clones was verified with a buffer control.

Techniques: Isolation, Emulsion, Recombinant, Incubation, Labeling, Flow Cytometry, Generated, Modification, Binding Assay, Clinical Proteomics, Confocal Microscopy, Staining

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Mouse immunization and analysis scheme. Bone marrow plasma cells (CD138 + ) were magnetically enriched and then used in our workflow. RBD-specific plasma cells were sorted with fluorescently labeled RBD-streptavidin tetramers. b , Sorting of RBD-specific mouse plasma cells. Cells were gated as live/CD138 + , and IgG-secreting RBD-specific plasma cells were sorted by FACS. The plots show 79,629 (CD138 + ) and 1,623 (IgG + ) events at 2% contour level. c , Overview of antibody sequences of sorted plasma cells. In total, 54 paired heavy- and light-chain sequences were obtained. The pie chart shows the 21 observed HV and LV gene combinations (HV–LV). HV–LV pairings are colored by the HV gene. Combinations that were characterized are shown in darker shades, while combinations that were not expressed are shown in lighter shades. The three most expanded expressed HV–LV combinations are highlighted with their CDRH3 amino acid sequence and frequency. d , Summary of anti-RBD ELISA. The plot shows the EC 50 with an antibody concentration range of 0.0002–400 nM. Antibodies that did not bind RBD at 400 nM are shown at an arbitrary EC 50 of 1,000 nM (gray diamonds). e , Characteristics of mouse anti-SARS-CoV-2 RBD antibodies with neutralizing capacity—variable domain sequences (V and J genes), third complementarity-determining region amino acid sequences (CDR3), equilibrium dissociation constants ( K D ) and IC 50 against WT SARS-CoV-2. f , In-tandem epitope binning experiment with mRBD1 and mRBD2. g , Crystal structure of mRBD2 with SARS-CoV-2 RBD (PDB: 8BE1 ). Top left, the RBD (green) in complex with mRBD2 Fab fragment (purple and pink for light and heavy chains) is superimposed with RBD complexed with ACE2 (gray; PDB: 6M0J ), showing how the Fab fragment overlaps significantly with ACE2. The main figure shows details of the RBD loop (green carbon atoms) binding to the CDRs of the mRBD2 Fab fragment.

Article Snippet: For in-tandem assays of mouse antibodies, biotinylated RBD (10 μg ml −1 ) was immobilized on streptavidin BLI biosensors (Sartorius) for 300 s. The biosensors were then probed with 100 nM of the first antibody for 600 s followed by 100 nM of the second antibody for 300 s. Independent binding of both clones was verified with a buffer control.

Techniques: Clinical Proteomics, Labeling, Sequencing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Binding Assay

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Human anti-RBD antibody discovery. B cells were negatively selected from fresh or frozen PBMCs 7–9 days after the second BNT162b2 vaccine dose and then used in the workflow. RBD-specific ASCs were sorted with fluorescently labeled RBD-streptavidin tetramers. b , Sorting of RBD-specific human ASCs. Representative layout for sorting of IgG + /RBD + hydrogel beads obtained from the encapsulation of fresh B cells on d9 post vaccination. Hydrogel beads were gated as live/CD20 − /CD38 + /IgM − /IgG + . The plot shows 1,484 events at 2% contour level. c , Overview of sequences of human ASCs sorted with RBD. In total, 185 paired heavy- and light-chain sequences were obtained. The pie chart shows the observed HV–LV combination colored by the HV gene. HV–LV combinations that were expressed recombinantly are highlighted with their clone names and shown in a darker shade. d , Affinities ( K D ) of human anti-RBD antibodies. One antibody (gray diamond) did not show binding to RBD at the tested concentrations. e , Neutralization of WT and Omicron BA.1 SARS-CoV-2 by human anti-RBD antibodies. IC 50 was calculated from experiments performed in duplicate. Antibodies with no quantifiable neutralizing capacity at the highest concentration tested (100 μg ml −1 ) are shown at an arbitrary IC 50 of 1,000 μg ml −1 (gray diamonds). The ten antibodies with the lowest IC 50 against WT SARS-CoV-2 were also tested against Omicron BA.1 SARS-CoV-2. Antibodies neutralizing both variants are highlighted in red with their clone names. Neutralization of WT SARS-CoV-2 by the REGEN-COV (Ronapreve) mAb cocktail (casirivimab and imdevimab) was tested in the same assay (blue diamond). f , Sandwich epitope binning experiment with pairs of neutralizing human anti-RBD antibodies. In the network plot, the nodes show the antibody clones, the connections indicate pairwise blocking and the shaded areas indicate whether the antibodies belong to the same clonotype. Colors indicate the VH gene as in c .

Article Snippet: For in-tandem assays of mouse antibodies, biotinylated RBD (10 μg ml −1 ) was immobilized on streptavidin BLI biosensors (Sartorius) for 300 s. The biosensors were then probed with 100 nM of the first antibody for 600 s followed by 100 nM of the second antibody for 300 s. Independent binding of both clones was verified with a buffer control.

Techniques: Labeling, Encapsulation, Binding Assay, Neutralization, Concentration Assay, Clone Assay, Blocking Assay

Journal: Nature Biotechnology

Article Title: Rapid discovery of monoclonal antibodies by microfluidics-enabled FACS of single pathogen-specific antibody-secreting cells

doi: 10.1038/s41587-024-02346-5

Figure Lengend Snippet: a , Human anti-S1 antibody discovery. B cells were negatively selected from PBMCs 7 days after the second BNT162b2 vaccine dose and then used in the workflow. RBD/S1-specific ASCs were sorted with fluorescently labeled RBD- and S1-streptavidin tetramers. b , Index sorting of S1-specific human ASCs. Layout for sorting of IgG- or IgA-secreting ASCs isolated on day 7 post vaccination with fluorescently labeled S1- and RBD-streptavidin tetramers. Based on index sorting, the ratio of the S1/RBD fluorescence signal was calculated for all sequenced cells, and four sequences that corresponded to events with the highest S1/RBD fluorescence signal ratio were selected for expression (highlighted as red squares). The plot shows 719 events at a 5% contour level, and the percentage of events in each window is indicated. c , Characteristics of human anti-S1 antibodies—variable domain genes (V and J), third complementarity-determining region amino acid sequences (CDR3), equilibrium dissociation constants ( K D ) and IC 50 against WT SARS-CoV-2. En dashes (–) denote that binding or inhibition was not quantifiable at the tested concentrations. d , Anti-S1 and anti-RBD ELISA of anti-S1 antibodies. An RBD-binding positive control (human IgG1κ, clone AM001414 ; BioLegend) is shown in black, and an isotype control (human IgG1κ, clone QA16A12; BioLegend) is shown in gray. The table shows the EC 50 with an antibody concentration range of 0.0002–400 nM. The plots show mean values ± s.d. of two independent experiments performed in duplicate.

Article Snippet: For in-tandem assays of mouse antibodies, biotinylated RBD (10 μg ml −1 ) was immobilized on streptavidin BLI biosensors (Sartorius) for 300 s. The biosensors were then probed with 100 nM of the first antibody for 600 s followed by 100 nM of the second antibody for 300 s. Independent binding of both clones was verified with a buffer control.

Techniques: Labeling, Isolation, Fluorescence, Expressing, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Positive Control, Control, Concentration Assay